Rabies Virus Vaccine Research Articles

In Vivo Safety Studies With SPBN GASGAS in the Frame of Oral Vaccination of Foxes and Raccoon Dogs Against Rabies.

In order to obtain Marketing Authorization for an oral rabies vaccine in the European Union, not only safety studies in the target species, red fox and raccoon dog, are required. Since baits are distributed unsupervised in the environment, specific safety studies in selected non-target species are compulsory. Furthermore, oral rabies vaccines are based on live, replication-competent viruses and thus distinct safety studies in the target species for such type of vaccines are also mandatory. Here, the results of these safety studies in target and selected non-target species for a 3rd generation oral rabies virus vaccine construct, SPBN GASGAS (Rabitec), are presented. The studies included the following species; red fox, raccoon dog, domestic dog, domestic cat, domestic pig, wild rodents. The following safety topics were investigated; overdose, repeated dose, dissemination, shedding, horizontal and vertical transmission. It was shown that SPBN GASGAS did not cause disease or any other adverse reaction in vaccinated animals and naïve contact animals. The vaccine did not disseminate within the host beyond the site of entry. No horizontal transmission was observed in wild rodents. In the target species, there was evidence that in a few cases horizontal transmission of vaccine virus could have occurred under these experimental conditions; most likely immediately after vaccine administration. The vaccine construct SPBN GASGAS meets therefore the latest revised minimal safety requirements as laid down in the European Pharmacopoeia.

Safety studies with the oral rabies virus vaccine strain SPBN GASGAS in the small Indian mongoose (Herpestes auropunctatus)

BackgroundOral vaccination of the small Indian mongoose against rabies has been suggested as a potential tool to eliminate mongoose-mediated rabies on several Caribbean islands. A recently developed oral rabies virus vaccine strain, SPBN GASGAS, has already been shown to be efficacious in this reservoir species. Since, all available oral rabies vaccines are based on replication-competent viruses and vaccine baits are distributed unsupervised in the environment, enhanced safety standards for such vaccine types are required.ResultsThe results of safety studies, including overdose, repeated doses, dissemination and different routes of administration, in the target species are presented. It was shown that the construct was apathogenic, irrespective of dose and route of administration. Even when it was inoculated directly in the brain, it did not induce rabies infection. Furthermore, the vaccine strain did not spread within the target species after direct oral instillation beyond the site of entry.ConclusionThe vaccine strain SPBN GASGAS meets the safety requirements for live rabies virus vaccines in this target species, the small Indian mongoose.

An assessment of shedding with the oral rabies virus vaccine strain SPBN GASGAS in target and non-target species

A safety requirement for live vaccines is investigating possible shedding in recipients since the presence of replication competent vaccine in secretions could result in direct and indirect horizontal transmission. This is especially relevant for oral rabies vaccine baits that are deliberately distributed into the environment. In the current study, survival of an oral rabies virus vaccine, SPBN GASGAS, was examined in excretions from different target and non-target species; red fox, raccoon dog, small Indian mongoose, raccoon, striped skunk, domestic dog, domestic cat and domestic pig. Saliva – and (pooled) fecal samples collected at different time points after oral administration of the vaccine strain were examined for the presence of viral RNA (rt-PCR). All PCR-positive and a subset of PCR-negative samples were subsequently investigated for the presence of infectious virus by isolation in cell culture (RTCIT). Up to 7 days post vaccine administration viral RNA could be detected in 50 of 758 fecal samples but no infectious virus was detected in any of the examined PCR-positive fecal samples. In contrast, RNA-fragments were detected in 248 of 1053 saliva swabs for an extended period (up to 10 days) after vaccine administration, but viable virus was only present during the first hours post vaccine administration in 38 samples. No infectious vaccine virus was isolated in saliva swabs taken 24 h or more after vaccine administration. Hence, no active shedding of the vaccine virus SPBN GASGAS after oral administration occurred and the virus isolated during the initial hours was material originally administered and not a result of virus replication within the host. Thus, potential horizontal transmission of this vaccine virus is limited to a short period directly after vaccine bait uptake. It can be concluded that the environmental risks associated with shedding after distributing vaccine baits containing SPBN GASGAS are negligible.

A thermostable messenger RNA based vaccine against rabies.

Although effective rabies virus vaccines have been existing for decades, each year, rabies virus infections still cause around 50.000 fatalities worldwide. Most of these cases occur in developing countries, where these vaccines are not available. The reasons for this are the prohibitive high costs of cell culture or egg grown rabies virus vaccines and the lack of a functional cold chain in many regions in which rabies virus is endemic. Here, we describe the excellent temperature resistance of a non-replicating mRNA based rabies virus vaccine encoding the rabies virus glycoprotein (RABV-G). Prolonged storage of the vaccine from -80°C to up to +70°C for several months did not impact the protective capacity of the mRNA vaccine. Efficacy after storage was demonstrated by the induction of rabies specific virus neutralizing antibodies and protection in mice against lethal rabies infection. Moreover, storing the vaccine at oscillating temperatures between +4° and +56°C for 20 cycles in order to simulate interruptions of the cold chain during vaccine transport, did not affect the vaccine’s immunogenicity and protective characteristics, indicating that maintenance of a cold chain is not essential for this vaccine.

Immunogenic response in obese patients undergoing rabies post-exposure prophylaxis with combined equine rabies immunoglobulin and rabies vaccination

BackgroundObesity is a risk factor for increased morbidity and mortality associated with many vaccine preventable infectious diseases such as influenza. Moreover, higher volume of passive rabies immunoglobulin (RIG) due to weight based dosing might suppress vaccine-induced immune responses in obese patients. This study aimed to evaluate the effect of obesity on humoral immune responses to combined equine RIG and rabies vaccine treatment among patients with WHO category III exposure to a rabies suspected animal. MethodsA single centre, prospective, open-labelled study among WHO category III rabies exposed patients was conducted to compare serum rabies virus neutralizing antibody (RVNA) responses measured by rapid fluorescent focus inhibition test between obese (body mass index, BMI > 30 kg/m2) and control (BMI < 25 kg/m2) patients after combined immunization with equine rabies immunoglobulin and purified chick-embryo cell rabies vaccine for post exposure prophylaxis treatment. ResultsPost-vaccination geometric mean titer (GMT) of RVNA concentrations between two groups at day 7 were 0.33 (95% CI: 0.23, 0.46) vs 0.39 (95% CI: 0.27, 0.55), 4.61 (95% CI: 3.20, 6.63) vs 3.78 (95% CI: 2.77, 5.16) at day 14, and 7.45 (95% CI: 5.86, 9.49) vs 5.93 (95%CI: 4.46–7.90) at day 28 for obese and control patients, respectively. There was no statistically significant difference of RVNA GMT between two groups. Seroconversion to at least adequate concentration (RVNA titer ≥0.5 IU/mL) rates were 34% at day 7 and 100% at days 14 and 28 in both groups. There were no immediate hypersensitivity reaction and no serious adverse events observed during the study period. ConclusionsThere was no evidence of immunosuppression of antibodies’ responses in obese patients. Combined ERIG and rabies virus vaccination for post exposure treatment is safe.

Efficacy of the oral rabies virus vaccine strain SPBN GASGAS in foxes and raccoon dogs

To test the immunogenicity and efficacy of a new oral rabies virus vaccine strain SPBN GASGAS in wildlife target species, one group of foxes and two groups of raccoon dogs were offered a bait containing 1.7 ml of the vaccine (106.6 FFU/ml; 106.8 FFU/dose) and subsequently challenged approximately 180 days later with a fox rabies virus isolate. One group of raccoon dogs (n=30) received the same challenge dose (100.7 MICLD50/ml) as the red foxes (n=29). The other group with raccoon dogs (n=28) together with 8 animals that received the vaccine dose by direct instillation into the oral cavity (DIOC) were infected with a 40-fold higher dose of the challenge virus (102.3 MICLD50/ml). All but one of the 29 vaccinated foxes survived the challenge infection; meanwhile all 12 control foxes succumbed to rabies. Twenty-eight of 30 vaccinated raccoon dogs challenged with the same dose survived the infection, however only six of 12 control animals succumbed. When the higher challenge dose was administered, all 12 control animals died from rabies and all 36 vaccinated animals (28 baited plus 8 DIOC) survived. Blood samples were collected at different time points post vaccination and examined by both RFFIT and ELISA. The kinetics of the measured immune response was similar for both species, although in RFFIT slightly higher values were observed in foxes than in raccoon dogs. However, the immune response as measured in ELISA was identical for both species. The oral rabies virus vaccine SPBN GASGAS meets the efficacy requirements for live rabies virus vaccines as laid down by the European Pharmacopoeia.

Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine.

Lagos bat virus (LBV) is a phylogroup II lyssavirus exclusively found in Africa. Previous studies indicated that this virus is lethal to mice after intracranial and intramuscular inoculation. The antigenic composition of LBV differs substantially from that of rabies virus (RABV) and current rabies vaccines do not provide cross protection against phylogroup II lyssaviruses. To investigate the potential role of the LBV matrix protein (M) and glycoprotein (G) in pathogenesis, reverse genetics technology was used to construct recombinant viruses. The genes encoding the glycoprotein, or the matrix and glycoprotein of the attenuated RABV strain SPBN, were replaced with those of LBV resulting in SPBN-LBVG and SPBN-LBVM-LBVG, respectively. To evaluate the immunogenicity of the LBV G, the recombinant RABV SPBNGAS-LBVG-GAS was constructed with the LBV G inserted between two mutated RABV G genes (termed GAS). All the recombinant viruses were lethal to mice after intracranial (i.c.) inoculation although the pathogenicity of SPBNGAS-LBVG-GAS was lower compared to the other recombinant viruses. Following intramuscular (i.m.) inoculation, only SPBN-LBVM-LBVG was lethal to mice, indicating that both the M and G of LBV play a role in the pathogenesis. Most interestingly, serum collected from mice that were inoculated i.m. with SPBNGAS-LBVG-GAS neutralized phylogroup I and II lyssaviruses including RABV, Duvenhage virus (DUVV), LBV, and Mokola virus (MOKV), indicating that this recombinant virus has potential to be developed as a pan-lyssavirus vaccine.

Prokaryotic Expression and Monoclonal Antibody Preparation of Rabies Virus Phosphoprotein

Background: Rabies is a zoonotic infectious disease that infects the human and animal central nervous system. Worldwide, especially in low-income countries, this disease is still a burden for public health. Among the rabies virus proteins, phosphoprotein plays a very important role in viral infection, and this research found that immunization of rabies virus vaccines could widely induce antibody responses against phosphoprotein, therefore rabies virus phosphoprotein may be a useful target for development of rapid and low cost serological diagnosis test, and therapeutic drugs. Objectives: The aim of this study was to prepare anti-rabies virus phosphoprotein monoclonal antibody, which is used for rapid detection and diagnosis of rabies virus infection. Methods: The phosphoprotein gene was amplified by the polymerase chain reaction (PCR), and sub-cloned in a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-RABV-P. Recombinant RABV-P was induced by isopropyl-β-D-Thiogalactopyranoside (IPTG), and then purified by the Ni-NTA purification system. Immunization of BALB/c mice with the recombinant protein was performed, and the spleen cells of the immunized mice and SP2/0 myeloma cells were fused together to obtain the monoclonal cell strains, and then identification of the characteristics of the antibody by the enzyme linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay was done. Results: The prokaryotic expression vector of pET-32a-RABV-P was successfully constructed. The fusion protein was expressed in Escherichia coli Rosetta and purified. After cell fusion, a hybridoma cell line 1A4 was successfully obtained. The antibody titer of the anti-RABV-P ascites reached 256,000. The results of western blotting and indirect immunofluorescence assay indicated that 1A4 hybridoma cell line was able to produce specific antibodies against rabies virus phosphoprotein. Conclusions: Recombinant rabies virus phosphoprotein could be successfully expressed in E. coli. A specific monoclonal antibody against rabies virus phosphoprotein has been successfully prepared.

Oral vaccination of wildlife against rabies: Differences among host species in vaccine uptake efficiency

Oral vaccination using attenuated and recombinant rabies vaccines has been proven a powerful tool to combat rabies in wildlife. However, clear differences have been observed in vaccine titers needed to induce a protective immune response against rabies after oral vaccination in different reservoir species. The mechanisms contributing to the observed resistance against oral rabies vaccination in some species are not completely understood. Hence, the immunogenicity of the vaccine virus strain, SPBN GASGAS, was investigated in a species considered to be susceptible to oral rabies vaccination (red fox) and a species refractory to this route of administration (striped skunk). Additionally, the dissemination of the vaccine virus in the oral cavity was analyzed for these two species. It was shown that the palatine tonsils play a critical role in vaccine virus uptake. Main differences could be observed in palatine tonsil infection between both species, revealing a locally restricted dissemination of infected cells in foxes. The absence of virus infected cells in palatine tonsils of skunks suggests a less efficient uptake of or infection by vaccine virus which may lead to a reduced response to oral vaccination. Understanding the mechanisms of oral resistance to rabies virus vaccine absorption and primary replication may lead to the development of novel strategies to enhance vaccine efficacy in problematic species like the striped skunk.

APRIL:TACI axis is dispensable for the immune response to rabies vaccination

There is significant need to develop a single-dose rabies vaccine to replace the current multi-dose rabies vaccine regimen and eliminate the requirement for rabies immune globulin in post-exposure settings. To accomplish this goal, rabies virus (RABV)-based vaccines must rapidly activate B cells to secrete antibodies which neutralize pathogenic RABV before it enters the CNS. Increased understanding of how B cells effectively respond to RABV-based vaccines may improve efforts to simplify post-exposure prophylaxis (PEP) regimens. Several studies have successfully employed the TNF family cytokine a proliferation-inducing ligand (APRIL) as a vaccine adjuvant. APRIL binds to the receptors TACI and B cell maturation antigen (BCMA)–expressed by B cells in various stages of maturation–with high affinity. We discovered that RABV-infected primary murine B cells upregulate APRIL ex vivo. Cytokines present at the time of antigen exposure affect the outcome of vaccination by influencing T and B cell activation and GC formation. Therefore, we hypothesized that the presence of APRIL at the time of RABV-based vaccine antigen exposure would support the generation of protective antibodies against RABV glycoprotein (G). In an effort to improve the response to RABV vaccination, we constructed and characterized a live recombinant RABV-based vaccine vector which expresses murine APRIL (rRABV-APRIL). Immunogenicity testing in mice demonstrated that expressing APRIL from the RABV genome does not impact the primary antibody response against RABV G compared to RABV alone. In order to evaluate the necessity of APRIL for the response to rabies vaccination, we compared the responses of APRIL-deficient and wild-type mice to immunization with rRABV. APRIL deficiency does not affect the primary antibody response to vaccination. Furthermore, APRIL expression by the vaccine did not improve the generation of long-lived antibody-secreting plasma cells (PCs) as serum antibody levels were equivalent in response to rRABV-APRIL and the vector eight weeks after immunization. Moreover, APRIL is dispensable for the long-lived antibody-secreting PC response to rRABV vaccination as anti-RABV G IgG levels were similar in APRIL-deficient and wild-type mice six months after vaccination. Mice lacking the APRIL receptor TACI demonstrated primary anti-RABV G antibody responses similar to wild-type mice following immunization with the vaccine vector indicating that this response is independent of TACI-mediated signals. Collectively, our findings demonstrate that APRIL and associated TACI signaling is dispensable for the immune response to RABV-based vaccination.

Inactivated Recombinant Rabies Viruses Displaying Canine Distemper Virus Glycoproteins Induce Protective Immunity against Both Pathogens.

The development of multivalent vaccines is an attractive methodology for the simultaneous prevention of several infectious diseases in vulnerable populations. Both canine distemper virus (CDV) and rabies virus (RABV) cause lethal disease in wild and domestic carnivores. While RABV vaccines are inactivated, the live-attenuated CDV vaccines retain residual virulence for highly susceptible wildlife species. In this study, we developed recombinant bivalent vaccine candidates based on recombinant vaccine strain rabies virus particles, which concurrently display the protective CDV and RABV glycoprotein antigens. The recombinant viruses replicated to near-wild-type titers, and the heterologous glycoproteins were efficiently expressed and incorporated in the viral particles. Immunization of ferrets with beta-propiolactone-inactivated recombinant virus particles elicited protective RABV antibody titers, and animals immunized with a combination of CDV attachment protein- and fusion protein-expressing recombinant viruses were protected from lethal CDV challenge. However, animals that were immunized with only a RABV expressing the attachment protein of CDV vaccine strain Onderstepoort succumbed to infection with a more recent wild-type strain, indicating that immune responses to the more conserved fusion protein contribute to protection against heterologous CDV strains.IMPORTANCE Rabies virus and canine distemper virus (CDV) cause high mortality rates and death in many carnivores. While rabies vaccines are inactivated and thus have an excellent safety profile and high stability, live-attenuated CDV vaccines can retain residual virulence in highly susceptible species. Here we generated recombinant inactivated rabies viruses that carry one of the CDV glycoproteins on their surface. Ferrets immunized twice with a mix of recombinant rabies viruses carrying the CDV fusion and attachment glycoproteins were protected from lethal CDV challenge, whereas all animals that received recombinant rabies viruses carrying only the CDV attachment protein according to the same immunization scheme died. Irrespective of the CDV antigens used, all animals developed protective titers against rabies virus, illustrating that a bivalent rabies virus-based vaccine against CDV induces protective immune responses against both pathogens.

Targeting Vaccine-Induced Extrafollicular Pathway of B Cell Differentiation Improves Rabies Postexposure Prophylaxis

Vaccine-induced B cells differentiate along two pathways. The follicular pathway gives rise to germinal centers (GCs) that can take weeks to fully develop. The extrafollicular pathway gives rise to short-lived plasma cells (PCs) that can rapidly secrete protective antibodies within days of vaccination. Rabies virus (RABV) postexposure prophylaxis (PEP) requires rapid vaccine-induced humoral immunity for protection. Therefore, we hypothesized that targeting extrafollicular B cell responses for activation would improve the speed and magnitude of RABV PEP. To test this hypothesis, we constructed, recovered, and characterized a recombinant RABV-based vaccine expressing murine B cell activating factor (BAFF) (rRABV-mBAFF). BAFF is an ideal molecule to improve early pathways of B cell activation, as it links innate and adaptive immunity, promoting potent B cell responses. Indeed, rRABV-mBAFF induced a faster, higher antibody response in mice and enhanced survivorship in PEP settings compared to rRABV. Interestingly, rRABV-mBAFF and rRABV induced equivalent numbers of GC B cells, suggesting that rRABV-mBAFF augmented the extrafollicular B cell pathway. To confirm that rRABV-mBAFF modulated the extrafollicular pathway, we used a signaling lymphocytic activation molecule (SLAM)-associated protein (SAP)-deficient mouse model. In response to antigen, SAP-deficient mice form extrafollicular B cell responses but do not generate GCs. rRABV-mBAFF induced similar anti-RABV antibody responses in SAP-deficient and wild-type mice, demonstrating that BAFF modulated immunity through the extrafollicular and not the GC B cell pathway. Collectively, strategies that manipulate pathways of B cell activation may facilitate the development of a single-dose RABV vaccine that replaces current complicated and costly RABV PEP.IMPORTANCE Effective RABV PEP is currently resource- and cost-prohibitive in regions of the world where RABV is most prevalent. In order to diminish the requirements for rabies immunoglobulin (RIG) and multiple vaccinations for effective prevention of clinical rabies, a more rapidly protective vaccine is needed. This work presents a successful approach to rapidly generate antibody-secreting PCs in response to vaccination by targeting the extrafollicular B cell pathway. We demonstrate that the improved early antibody responses induced by rRABV-mBAFF confer improved protection against RABV in a PEP model. Significantly, activation of the early extrafollicular B cell pathway, such as that demonstrated here, could improve the efficacy of vaccines targeting other pathogens against which rapid protection would decrease morbidity and mortality.

Rabies virus vaccine as an immune adjuvant against cancers and glioblastoma: new studies may resurrect a neglected potential.

To review the literature about the use of Rabies Virus-Vaccine (RV-V) as an anticancer immunotherapeutic modality in the light of recent findings. The literature search in relevant databases with the following key words: Rabies virus, cancer, remission. Remissions occured following RV-V injections in patients with cervical cancer and melanoma. Pilot clinical studies showed that RV-V injections enhanced survival in glioblastoma patients, which is supported by findings in GL261 mouse glioma model. If public health studies demonstrate protective role of RV-V against certain types of cancers, it can be benefitted as a novel immune adjuvant in clinic.

Monitoring of rabies in wild animals in the Kirov region after oral immunization.

This work presents the results of the molecular genetic research on genomes of field isolates of the rabies virus circulating in the territory of the Kirov region in order to analyze the phylogenetic relationship between the wild isolate genomes and to determine the possible reversion of the vaccine strain of the rabies virus used in the oral vaccine to virulent variant. We studied 24 brain samples from wild carnivores shot after oral immunization of the area with Rabivak-O/333. A bait with the vaccine provided by the Veterinary Service of the Kirov was also studied. All samples were found to be positive for the presence of the rabies virus as established by FAT and RT-PCR techniques. Phylogenetic analysis of N genome fragments of the rabies virus showed that the field isolates from the Kirov regions were genetically close to the field isolates from Buryatia 2012. Analysis of G genome fragments showed that the Kirov field isolates were close to the isolates from Lipetsk (2011), as well as to the Ukrainian isolates (2006 and 2010). Molecular genetic analysis of the gene fragments N and G for the field isolates and fragments of the genome of the rabies virus vaccine did not reveal any reversion to the virulent vaccine strain.

Vaccine-induced rabies case in a cow (Bos taurus): Molecular characterisation of vaccine strain in brain tissue

Rabies is a fatal neuropathogenic zoonosis caused by the rabies virus of the Lyssavirus genus, Rhabdoviridae family. The oral vaccination of foxes — the main reservoir of rabies in Europe — using a live attenuated rabies virus vaccine was successfully conducted in many Western European countries. In July 2015, a rabies vaccine strain was isolated from the brain tissues of a clinically suspect cow (Bos taurus) in Romania. The nucleotide analysis of both N and G gene sequences showed 100% identity between the rabid animal, the GenBank reference SAD B19 strain and five rabies vaccine batches used for the national oral vaccination campaign targeting foxes.

Isatis indigotica root polysaccharides as adjuvants for an inactivated rabies virus vaccine

Adjuvants can enhance vaccine immunogenicity and induce long-term enhancement of immune responses. Thus, adjuvants are important for vaccine research. Polysaccharides isolated from select Chinese herbs have been demonstrated to possess various beneficial functions and excellent adjuvant abilities. In the present study, the polysaccharides IIP-A-1 and IIP-2 were isolated from Isatis indigotica root and compared with the common vaccine adjuvant aluminum hydroxide via intramuscular co-administration of inactivated rabies virus rCVS-11-G into mice. Blood was collected to determine virus neutralizing antibody (VNA) titers and B and T lymphocyte activation status. Inguinal lymph node samples were collected and used to measure B lymphocyte proliferation. Splenocytes were isolated, from which antigen-specific cellular immune responses were detected via ELISpot, ELISA and intracellular cytokine staining. The results revealed that both types of polysaccharides induce more rapid changes and higher VNA titers than aluminum hydroxide. Flow cytometry assays revealed that the polysaccharides activated more B lymphocytes in the lymph nodes and more B and T lymphocytes in the blood than aluminum hydroxide. Antigen-specific cellular immune responses showed that IIP-2 strongly induced T lymphocyte proliferation in the spleen and high levels of cytokine secretion from splenocytes, whereas aluminum hydroxide induced proliferation in only a small number of lymphocytes and the secretion of only small quantities of cytokines. Collectively, these data suggest that the polysaccharide IIP-2 exhibits excellent adjuvant activity and can enhance both cellular and humoral immunity.

Use of Rabies Virus Vaccine As a Neoantigen to Evaluate Humoral Immune Function in Patients with Primary Immunodeficiency Disorders Receiving Immunoglobulin Replacement Therapy

Use of Rabies Virus Vaccine As a Neoantigen to Evaluate Humoral Immune Function in Patients with Primary Immunodeficiency Disorders Receiving Immunoglobulin Replacement Therapy

A Novel Cell Substrate Candidate for Rabies Virus Vaccine Propagation and Production

Rabies is acute and fatal viral encephalitis and is estimated to cause more than 50000 human deaths annually. Several cell cultures have been introduced for rabies vaccine production such as human diploid (HDC), primary hamster kidney, fetal bovine kidney, chick embryo, and continuous line of monkey kidney (VERO) cells. In this study, a novel cell line, derived from dental pulp stem cells (RHDP) was used for rabies virus propagation and was compared to MRC-5 cell line. MRC5 showed a density higher than RHDP. The total cells obtained in a 25ml flask, was 4.2x10 6 ± 1.5x10 5 and 2.1x10 6 ± 2.6x10 5 for MRC-5 and RHDP, respectively. However, RHDP reached its maximum density sooner than MRC. The maximum cell specific growth rate of RHDP was 0.034 μ (h-1) while that of MRC was only 0.010 μ (h-1). The maximum cell division number of RHDP was 1.53 which was achieved three days after culture. Adaptation of the virus to the novel RHDP cell line was achieved after six passages of sub-culturing of the virus every 3-4 days. Rabies virus caused typical CPE and RHDP-cell-lysis in monolayers which were easily observed by microscopy. Our results suggest that RHDP could be another cell substrate candidate for rabies virus titration. The maximum specific virus production in RHDP and MRC-5 were 5.243 FFU/cell/h and 3.625 FFU/cell/h respectively; both had lower titers than those reported for Vero cell.

Commentary: Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs.

Rabies virus is accountable for substantial economic burden, and possesses high risk to the individuals living in low-income countries. Rabies is fatal encephalitis caused by the rabies virus (RABV) and usually transmit to humans through bites from rabid animals. The causative agent for this life threatening disease is RABV, containing single-stranded negative-sense RNA as genetic material (Jackson, 2013). Annually, rabies is responsible for approximately 55,000 deaths worldwide (a substantial part of deaths occur in developing countries); (Ge et al., 2011). Vaccination is the principal path for prevention and control of the disease. Currently available cell culture based rabies vaccines possess several side effects (e.g. fever, headache, neurological disease etc.) and high cost in developing countries usually require many doses to provide pre and post-exposure protection against the RABV. Several, improved vaccines based on anti-rabies monoclonal antibodies, lyophilized, and cell culture technology, are already under clinical development phase (Kaur et al., 2015). Furthermore, recombinant molecules have also been used as vaccine candidates very extensively. Further, immunogenicity of these molecules can be enhanced by combining adjuvants and immuno-stimulatory molecules. In the past, Wen et al. demonstrated that recombinant rabies virus (rRABV) expressing dendritic cell activating molecules such as granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein (MIP-1a), could enhance RABV immunogenicity (Wen et al., 2011). Moreover, Zhou et al. suggested that rRABV expressing bacterial flagellin could also induce robust adaptive immune responses (Zhou et al., 2013). Flagellin is a very effective and potent adjuvant because of non-inducer of IgE production and at very low dose (1–10 μg in non-human primates) very efficient to induce effective immune responses (Mizel and Bates, 2010). Recombinant virus-like particles (VLPs) are promising vaccine candidates, because they mimic original virus structure and are able to induce potent humoral and cellular immune responses (Kumar et al., 2011). Moreover, VLP possesses several advantages over the conventional vaccines because of the lack of genetic material, stable in extreme environment and capable of expressing foreign molecules. Very recently, Fontana et al. (2014) showed that VLPs containing glycoprotein of RABV are very efficient in induction of antibody mediated immune responses in Balb/c mice. Qi et al. (2015) combined all these novel strategies, in order to develop an effective RABV vaccine. In present report, Qi and colleagues constructed chimeric RABV-like particles (cRVLPs) containing RABV glycoprotein and matrix protein, expressing membrane-anchored flagellin or E. coli heat-labile enterotoxin B (LTB). Immunological studies in BALB/c mice and dog models demonstrated that both humoral and cellular immune responses were stronger with cRVLPs, compared with standard rabies VLPs (sVLPs). Further, Qi et al. (2015) suggest that flagellin promote antigen processing and presentation by inducing higher expression of MHC I and II on the surface of DCs. Interestingly, in a pioneer study, mucosal delivery of inactivated influenza vaccine have been shown to induce heterotypic immunity against H1N1 virus, when administered with heat-labile enterotoxin B (Tumpey et al., 2001). These attributes make flagellin and enterotoxin B outstanding adjuvants for use in vaccines. In conclusion, present and previous reports suggest that chimeric VLPs are novel class of subunit vaccines and possess capability to induce both protective innate and adaptive immune responses. Possibly, use of immuno-modulators along with cRVLPS may enhance the efficacy of existing rabies vaccines. Further, detailed mechanism of the comprehensive effect of flagellin and enterotoxin B stimulated T and B-cells, on antiviral immune responses, should be investigated.

Biotechnology Dept., Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Nadi El-Seid St., P.O. Box 246, Dokki, Giza 12618, Egypt

To improve the efficacy of the inactivated rabies virus vaccine and vaccination of dogs and cats against rabies virus disease, ginsenoside Re was extracted from Panax ginsing used as adjuvant to rabies vaccine in different concentrations: 1.25, 2.50 and 5.00 mg/kg wt. This work included dogs and cats divided into eight groups of which five groups were vaccinated with inactivated rabies virus vaccine adjuvanted by ginsenoside Re. The 6th group was vaccinated with inactivated rabies vaccine adjuvanted by aluminum hydroxide gel (20%). On the other hand, the 7th group was inoculated by ginsenoside Re subcutaneously and the 8th group was left unvaccinated as control. It was revealed that ginsenoside Re is safe and did not show any clinical signs. Estimation of humoral immune response of vaccinated animals via indirect ELISA and serum neutralization test (SNT) revealed that vaccinated animals acquired rabies antibodies with high protective level by using ginsenoside Re as adjuvant. The obtained results were compared with those induced by the traditional gel vaccine. It was found that the use of Ginsenoside-Re could reduce the required protective dose and subsequently increase production rate. From these results, it could be concluded that ginsenoside-Re could be used as adjuvant to inactivated rabies virus vaccine.